Title

Author

Defense Date

2009

Document Type

Thesis

Degree Name

Master of Science

Department

Biochemistry

First Advisor

Paul Ratz

Abstract

KCl is traditionally used as a stimulus to examine the role of increases in cytosolic Ca2+ on the regulation of smooth muscle contraction. KCl bypasses GPCR activation, thereby avoiding activation of additional cell signaling systems, such as phospholipase C and PKC that are inherent to Gaq and Ga12/13 stimulation. GPCR activation causes Ca2+ sensitization (greater force for a given increase in Ca2+) by a ROCK- and PKC-dependent inhibition of myosin light chain (MLC) phosphatase. Recent studies have demonstrated that KCl can also produce Ca2+ sensitization, implying that Ca2+ itself may induce Ca2+ sensitization. To test the hypothesis that Ca2+ can induce Ca2+ sensitization, we permeabilized rabbit femoral artery rings with beta-escin and subjected the tissues to a Ca2+ concentration response curve in the absence (control) and presence of selective inhibitors of ROCK and PKC, the general ser/thr kinase inhibitor, staurosporine, and the MLCK inhibitor, wortmannin. For a comparison, tissues permeabilized with beta-escin were also contracted with the alpha-adrenergic agonist, phenylephrine (PE), and exposed to the same complement of inhibitors. Interestingly, Ca2+-induced contraction was inhibited by a PKC inhibitor in beta-escin-permeabilized tissue. Wortmannin and staurosporine nearly abolished Ca2+-induced contraction. The ROCK inhibitor, H-1152, significantly reduced Ca2+- and PE-induced contractions in beta-escin permeabilized tissues. Western blots showed significant decreases in basal MYPT1-pThr853 by ROCK inhibitors and reduction of MLC-p by ROCK and PKC inhibitors. These data support our hypothesis that elevations in Ca2+ or constitutive ROCK activity may cause ROCK-dependent Ca2+ sensitization. Moreover, data also suggest the possibility of PKC-directed activation of the RhoA/ROCK cascade. Once the mechanism for smooth muscle Ca2+ sensitization is understood, advanced treatments for vascular hyper-contraction disorders, such as vasospasm and hypertension, may be possible.